The present invention relates to a method for designing and preparing mutant transglutaminase on the basis of the three-dimensional structure of transglutaminase derived from Streptoverticillium mobaraense (hereinafter referred to as “MTG”) determined by X-ray crystal structure analysis techniques, and the mutant MTGs thus prepared. MTG is widely used for processing foods or the like because it forms a gelatinus substance by forming crosslinking between proteins. The mutant MTG improved in the transglutaminase activity and heat stability contributes to reduce the required amount thereof, and also the mutant MTG having modified substrate specificity and optimum pH will allow the application of the enzyme to a new field.
Transglutaminase is an enzyme which catalyzes the acyl transfer reaction of γ-carboxamide group existing in a peptide chain of a protein. By reacting a protein with this enzyme, ε-(γ-Glu)-Lys crosslinking reaction and replacement reaction of Gln with Glu by deamidation may occur.
Transglutaminases from animals and those from microorganisms are so far known. The former is a Ca2+-dependent enzyme and it is widely distributed in organs, skin and blood of animals. This includes, for example, guinea pig liver transglutaminase [K. Ikura et al., Biochemistry, Vol. 27, p. 2898 (1988)], human epidermal keratin cell transglutaminase [M. A. Phillips et al., Proc. Natl. Acad. Sci., USA, Vol. 87, p. 9333 (1990)] and human blood coagulation factor XIII [A. Ichinose et al., Biochemistry, Vol. 25, p. 6900 (1990)]. As for the latter, the Ca2+-independent ones were found in Streptoverticillium bacteria such as Streptoverticillium griseocarneum IFO 12776, Streptoverticillium cinnamoneum sub sp. cinnamoneum IFO 12852 and Streptoverticillium mobaraense IFO 13819. Among these enzymes, the transglutaminase found in a culture supernatant of a variant of Streptoverticillium mobaraense is referred to as MTG (Microbial Transglutaminase). Also, a Ca2+-independent transglutaminase was found in Streptomyces lydicus NRRL B-3446 (JP-Kokai No. 10-504721).
MTG is a monomeric protein composed of 331 amino acids and having a molecular weight of about 38,000 [Journal of Biological Chemistry, vol. 268, p. 11565 (1993)]. A method for producing active MTG by secretion expression using E. coli or yeast was reported (JP-Kokai No. Hei 5-199883). There has also been reported a method for producing the same wherein MTG is expressed as a protein inclusion body in E. coli, then the inclusion body is solubilized with a protein denaturing agent and it is then regenerate through removing the denaturing agent to obtain active MTG (JP-Kokai No. 6-30771).
Unlike transglutaminases from animals, the transglutaminases from microorganisms, such as MTG, are Ca2+-independent. They are therefore utilized for the production of gelled foods such as jellies, as well as yogurts, cheeses or cosmetics in gel form, and also for the improvement of the quality of meats (JP-Kokai No. 64-27471). They are highly useful in the industry in that they are used as materials for heat stable microcapsules or as carriers for immobilized enzymes.
As for the conditions of the enzymatic reaction, for example, gelatinized foods cannot be gelled when the enzymatic reaction time is short, on the contrary, they become too hard to be the commercial products when the enzymatic reaction time is excessively long. Therefore, when MTG is used for producing gelatinized foods such as jellies; yogurts; cheeses; or cosmetics in gel form; or for improving the quality of meats, the concentration of the substrate and enzyme, reaction temperature and reaction time are controlled suitably for the intended product. However, as the foods and reagents produced by using MTG became various, it occurred that the intended products could not be produced in some cases by only controlling the concentration, temperature, time, etc. Thus, the modification of the enzymatic activity of MTG has become necesssary.
For modifying the enzymatic activity of MTG, it is required to produce a MTG mutant and to evaluate the activity, substrate specificity, etc. of the mutant to find out a good mutant. For producing mutants, it is necessary to manipulate wild type genes and, accordingly, it is prerequisite that a recombinant protein can be prepared. For MTG, a large-scale expression system using E. coli has been established (JP-Kokai No. 6-30771). However, MTG thus expressed in a large amount in E. coli is accumulated as insoluble protein inclusion bodies in the microbial cells. The denatured MTG in the protein inclusion body must be activated by refolding it after solubilizing it. Thus, it takes 2 to 3 weeks to prepare one mutant MTG. It has therefore been required to develop a method for reasonably modify MTG using some technique other than the techniques of randomly introducing the mutations through the entire molecule.